Method for culturing cell and a culture vessel

ABSTRACT

A method for adhering and proliferating cell, which comprises the steps of inoculating, culturing and then killing fibroblast derived from a mammal, is provided. A culture vessel manufactured according to the steps of the method which can provide improved adhesion to cell and enhanced cell-proliferation is also provided.

BACKGROUND OF THE INVENTION

The present invention relates to a method for adhering and proliferatingcell. Particularly, the present invention relates to a method foradhering and proliferating epithelial and hepatic cell. The presentinvention also relates to a method for culturing epidermal cell to beused in an epidermal cell sheet and an epidermal cell suspension whichcan be applied to an apellous part such as those with burn, wound,bedsore or skin ulcer for early reconstruction or treatment of suchdamaged tissue, an epidermal cell sheet and suspension prepared by theculture method, and a method for culturing hepatic cell which isimportant in analysis of hepatic function.

The present invention also relates to a culture vessel manufacturedaccording to the steps of the methods of the present invention, whichcan provide improved adhesion to cell and enhanced cell-proliferation.Particularly, the present invention relates to a culture vessel whichcan provide improved adhesion to epithelial and/or hepatic cell andenhanced epithelial and/or hepatic cell-proliferation. Moreparticularly, the present invention relates to a culture vessel thatenables preparation of an epidermal cell sheet and an epidermal cellsuspension to be applied to an apellous part such as those with burn,wound, bedsore or skin ulcer for early reconstruction or treatment ofsuch damaged tissue.

Conventionally, two methods have widely been employed for culturingepithelial cell, particularly epidermal cell (or which is calledepidermal keratinocyte). One utilizes sterilized 3T3 mouse embryofibroblast, i.e., viable 3T3 mouse embryo fibroblast from which divisionand proliferation potencies have been deleted by irradiating, forexample, γ ray or by adding an agent such as mitomycin C, as feederlayer (such as the feeder layer culture method described in James G.Rheinwald and Howard Green. Cell 6:331-344. Serial Cultivation ofStrains of Human Epidermal Keratinocytes: the Formation of KeratinizingColonies from Single Cells). The other utilizes serum-free medium suchas MCDB153 instead of feeder layer.

However, the conventional feeder layer culture method in which 3T3 mouseembryo fibroblasts are used as a feeder layer involves complicatedprocedure for preparing the 3T3 mouse embryo fibroblasts immediatelybefore epidermal cells are inoculated, and such feeder layer has alimited life time. During proliferation of epidermal cells other thanmouse epidermal cells such as human epidermal cells and preparation ofan epidermal cell sheet, those cells may possibly be contaminated withheterogenous cells, 3T3 mouse embryo fibroblasts, and an agent such asmitomycin C, which is added to delete division and proliferationpotencies of the 3T3 embryo fibroblasts, may remain.

On the other hand, when fibroblast homogenous to the epidermal celland/or epidermal cell sheet of interest is used as the feeder cellinstead of 3T3 mouse embryo fibroblast (e.g., human fibroblast may beused for preparing a human epidermal cell sheet), there is nopossibility of contamination with heterogenous cells. However, theirdivision and proliferation potencies should also be deleted by adding anagent such as mitomycin C that may possibly remain. Human fibroblast maybe used, but they provide slower proliferation rate of the epidermalcells when compared to that obtained by using 3T3 mouse embryofibroblast.

Culture method employing serum-free medium may often provide slowerproliferation rate of epidermal cell and require longer period of timefor incubation when compared to feeder layer culture methods whichemploy feeder cells such as 3T3 mouse embryo fibroblast. Further,serum-free medium is likely to suppress the differentiation of epidermalcell, which may cause inability of the epidermal cell to form amultiple-layer, resulting in unsuccessful preparation of an epidermalcell sheet.

Japanese Unexamined Patent Application Publication No. 285781/1987disclosed a method which employs a feeder layer to culture hepaticcells. This method may also have a possibility of residual agent orcontamination with heterogenous cells. Further, this method requirescontinual subculture of feeder cell to keep the cell ready for use underan optimal condition (subconfluent), making the procedure verycomplicated.

SUMMARY OF THE INVENTION

According to the investigation of various culture conditions forepidermal cell forming an epidermal cell sheet and hepatic cell, a novelmethod is found, which comprises the steps of inoculating, culturing andthen killing fibroblast derived from a mammal and separating the killedfibroblasts from the vessel at least partially to substantially leave acomponent or components, on the surface of the culture vessel, such asthe accumulated extracellular matrix which has been secreted from theculture cells. Thus, this method does not require preparing feeder cellssuch as fibroblasts nor sterilizing the cells as required inconventional feeder layer culture methods. Further, a culture vesselmanufactured according to the steps of the above method, which canprovide improved adhesion to cell and enhanced cell-proliferation, mayenable to be preserved for long time while keeping its property.Accordingly, the culture vessel does not require daily subculture (i.e.,transferring cells on another vessel in order to prevent overpopulationof the cells in the culture vessel) of fibroblasts to be used as thefeeder cells. Preservation of a large stock of the culture vessels canbe preserved in a cold and dark place. Further, the culture vessel whichcan provide improved adhesion to cell and enhanced cell-proliferationwhen compared to one manufactured by conventional methods can beobtained. On the basis of these findings, the present invention has beencompleted.

Accordingly, one object of the present invention is to provide a methodfor adhering and proliferating cell comprising the steps of inoculating,culturing and then killing fibroblast derived from a mammal, which canprovide improved cell adhesion and proliferation potencies when comparedto those provided by conventional feeder layer culture method, withoutpreparing feeder cells such as fibroblasts nor sterilizing such cells asrequired in conventional feeder layer culture methods, and thus canprovide an epidermal cell sheet, an epidermal cell suspension or hepaticcells while avoiding contamination with heterogenous cells. Anotherobject of the present invention is to provide the followings: a methodfor culturing epidermal cell to be used in an epidermal cell sheet andan epidermal cell suspension which can be applied to an apellous partsuch as those with burn, wound, bedsore or skin ulcer for earlyreconstruction or treatment of the damaged tissue; an epidermal cellsheet and an epidermal cell suspension prepared by the culture method;and a method for culturing hepatic cells which is important in analysisof hepatic function.

Still another object of the present invention is to provide a culturevessel which can provide improved adhesion to cell and enhancedcell-proliferation according to the steps of the above method.Particularly, one object of the present invention is to provide aculture vessel which can provide improved adhesion to cell and enhancedcell-proliferation, which are manufactured by culturing and killingfibroblasts derived from a mammal (particularly 3T3 mouse embryofibroblast) by, for example, freezing and/or drying in a culture vesseland separating the killed fibroblasts from the vessel at least partiallyto substantially leave a component or components such as the accumulatedextracellular matrix which has been secreted from the culture cells toremain on the surface of the culture vessel, i.e., to leave component(s)necessary for cell adhesion and proliferation to remain on the surfaceof the culture vessel.

In summary, the present invention relates to a method for adhering andproliferating cell which comprises the steps of inoculating, culturingand then killing fibroblast derived from a mammal.

In the method, killed fibroblast may be preferably separated from thevessel partially, and more preferably entirely.

In the method, fibroblast may be killed by freezing, drying and/orirradiating electromagnetic radiation or by repeating one treatmentselected from the group consisting of freezing, drying and irradiatingelectromagnetic radiation. Fibroblast may also be killed by acombination of at least two treatments selected from the groupconsisting of freezing, drying and irradiating electromagneticradiation. In the method, electromagnetic radiation may be at least oneselected from the group consisting of β ray, γ ray, X-ray, electron beamand UV ray.

In the method, 3T3 mouse embryo fibroblast may be used as fibroblastderived from a mammal.

In the method, epithelial or hepatic cell may be used as the cell whichadheres and proliferates. Epidermal cell is preferable as epithelialcell.

Further, the present invention relates to epidermal cell which has beencultured using the method. The present invention also relates to anepidermal cell sheet or suspension prepared from the epidermal cell.

The present invention also relates to a culture vessel manufacturedaccording to the steps of the method, which can provide improvedadhesion to epithelial and/or hepatic cell and enhanced epithelialand/or hepatic cell-proliferation.

In the culture vessel, killed fibroblast may be preferably separated atleast partially, or entirely.

Preferably, the culture vessel can be preserved by freezing and/ordrying.

For the culture vessel, 3T3 mouse embryo fibroblast is used asfibroblast derived from a mammal preferably.

For the culture vessel, epidermal keratinocyte may be used preferably asthe epithelial cell.

The material of the culture vessel may be glass, synthetic polymer orbiopolymer. Further, the culture vessel may have any shape such asflask, petri dish, roller bottle, tray, well plate, beads, film, sheetor sponge.

The culture vessel made of glass or synthetic polymer may have any shapesuch as flask, petri dish, roller bottle, tray, well plate, beads, film,sheet or sponge.

Preferably, the culture vessel made of biopolymer may have any shapesuch as sheet, film, sponge or beads.

The culture vessel may be used to prepare an epidermal cell sheet.

The culture vessel may be used to prepare an epidermal cell suspension.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 illustrates a bar graph showing the average number of epidermalcells collected from each of the culture flasks (culture surface: 25cm²) of frozen 3T3 and the control in Example 1 in triplicate byperforming the test in triplicate.

DETAILED DESCRIPTION

Cells to adhere and proliferate in the cell adhesion and proliferationmethod according to the present invention include those that can becultured by any conventional feeder layer culture method, such asepithelial cells and hepatic cell. The term “epithelial cell” hereinencompasses the followings: epithelial cells such as mucosal epithelialcells (which are surface cells of intestine, oral cavity or nasalcavity) and corneal epithelial cells; and epidermal cells which exist onskin epithelium and are denucleated and keratinized after cell division.Hepatic cells are cells that constitute a hepatic lobule.

For the cell adhesion and proliferation method according to the presentinvention, it is important to kill fibroblast derived from a mammalafter culturing them separate from the vessel in order to remove thekilled fibroblasts at least partially, and leave extracellular matrixrequired for cell adhesion and proliferation on the surface of theculture vessel.

Fibroblasts may be used those derived from mammals such as mouse, human,rat, hamster and rabbit. Preferably, 3T3 mouse embryo fibroblast, whichis commonly used in conventional feeder layer culture methods, may beused. The condition for inoculating and culturing cell is notparticularly limited, and any standard condition may be used. Forexample, fibroblasts grown in a culture vessel may be separated bytreating with trypsin solution (which was prepared by dissolving trypsin(0.25 weight/volume %) in a solution of 0.206 mg/mlethylenediamine-tetraacetic acid (EDTA) in phosphate buffer). Theseparated fibroblasts were suspended in a medium supplemented with 5 to10% fetal bovine serum, inoculated in the culture vessel, and then leftto stand in a CO₂ incubator. No special culture vessel, for example, aculture vessel coated with extracellular matrix such as collagen, isrequired. Any material or shape may be used for the culture vessel aslong as 3T3 fibroblasts, for example, can adhere to and proliferate inthe culture vessel. Any culture vessel for adhesive cell which arecommercially available such as flask, petri dish, roller bottle, wellplate or tray, or any carriers such as conventional synthetic polymermembrane, film or plate, or biopolymer membrane, film or microbeads maybe used, which can greatly reduce the process costs when compared to anyconventional methods.

An embodiment of culture vessel will be described which can provideimproved adhesion to cell and enhanced cell-proliferation according tothe steps of the cell adhesion and proliferation method which comprisesthe steps of inoculating, culturing and then killing fibroblast derivedfrom a mammal.

Fibroblasts are grown in a culture vessel and the grown fibroblasts arekilled. The killed fibroblasts (hereinafter referred to as “dead cells”)are then removed by separation, and component or components required forcell adhesion and proliferation are left on the surface of the culturevessel. As a result, property which can provide improved adhesion tocell and enhanced cell-proliferation will be rendered to the surface ofthe culture vessel. For the purpose of rendering such property to thesurface of the culture vessel, fibroblasts are inoculated preferably at1.0×10² to 1.0×10⁵ cells/cm², and more preferably at 3.0×10³ to 1.0×10⁵cells/cm², and then cultured under the above conditions.

In the subsequent step of killing the fibroblasts, it is important tokill substantially all the fibroblasts under the condition which may notdegenerate the component or components required for cell adhesion andproliferation. Killing substantially all the fibroblasts may facilitateand ensure the removal of the fibroblasts. Thus, contamination of theresultant epidermal cell sheet, epidermal cell suspension or hepaticcells with heterogenous cells can be prevented. Further, unlikeconventional sterilization such complete killing may not require addingan agent such as mitomycin C, thus avoiding possibility of residualagent.

Fibroblasts may be killed by any method that may not degenerate thecomponent or components required for cell adhesion and proliferation norinhibit the cell adhesion and proliferation. Such methods includefreezing, freeze-drying, drying, drying at low temperature, irradiationof electromagnetic radiation such as β ray, γ ray, X-ray, electron beamand UV ray, and a combination of such treatments. These methods canretain the property of the culture vessel which can provide improvedadhesion to cell and enhanced cell-proliferation. When epidermal cellsare inoculated and cultured in such a culture vessel, they may adhereand proliferate well to prepare an epidermal cell sheet for practicaluse.

Fibroblasts may be preferably killed by freezing and drying. Freezingmay be easily manipulated, and can kill a large number of fibroblasts atone time. Further, an apparatus to be used for freezing is generallycheaper than that used in other methods. After fibroblasts are killed,the culture device can be continually frozen for long-time preservationwhile retaining its property which can provide improved adhesion to celland enhanced cell-proliferation. Freezing treatment may comprisefreezing and thawing, or repeated freezer-thawing procedure. Freezingtreatment includes freezer-dryer, freezer, ultra-deep freezer, liquefiedCO₂ or liquefied nitrogen gas. Any freezing temperature may be usedwhich can freeze cells. Usually, 0° C. or lower may be used. Preferably,fibroblasts may be gradually frozen by using, for example, aprogrammable freezer since freezing rate may affect the death offibroblasts.

For drying treatment, any conventional dryer may be used. Dryingtemperature may be preferably from non-freezing temperature (around 0°C.) to 60° C., and more preferably 4° C. to 30° C. As described above,it is important to inhibit degeneration of active component(s).Therefore, fibroblasts may be desirably killed under these moderateconditions.

Fibroblasts may also be killed by irradiating electromagnetic radiationselected from the group consisting of electron beam, γ-ray and UV-ray.Preferable range of irradiating electron beam or γ-ray is 5 to 30 kGy,and preferable range of irradiating UV-ray is 50 to 5000 mW•sec/cm². Forthe purpose of rendering property which can provide improved adhesion tocell and enhanced cell-proliferation to the surface of the culturevessel, fibroblasts are inoculated preferably at 1.0×10² to 1.0×10⁵cells/cm², and more preferably at 1.0×10³ to 1.0×10⁵ cells/cm², and thencultured under the above conditions.

After culturing, culture supernatant of the fibroblast is removed, andthen the remainder is treated by irradiating 5 to 30 kGy electron beams.Before irradiating electron beam or γ-ray, i.e., after culturesupernatant of the fibroblast is removed, the culture vessel ispreferably kept at most 4° C. More preferably, a culture vessel istreated by irradiating ray after freezing treatment. Such conditions ofthe treatment prevent component or components required for cell adhesionand proliferation from degeneration. When a vessel is treated byirradiating electron beam keeping at 4° C., for example, adhesion tocell and cell-proliferation are a little inferior when compared tofeeder layer culture methods. However, an equivalent epidermal cellsheet can be prepared when compared to that obtained according to feederlayer culture methods. In the culture vessel which is treated byirradiating ray after freezing treatment, adhesion to cell andcell-proliferation are equal when compared to feeder layer culturemethods. Irradiating electron beam or γ-ray is useful to kill fibroblastcompletely and sterilize a culture vessel.

After fibroblasts are killed by the above treatment, dead cells areremoved by separation. The term “separate” or “separation” hereinintends to encompass all the manipulations for removing dead cells suchas detaching or washing.

In this process, fibroblasts should be completely removed in order toprevent heterogenous cells such as 3T3 mouse embryo fibroblast fromcontaminating the epidermal cell sheet, epidermal cell suspension orhepatic cells obtained by using the culture vessel manufacturedaccording to the steps of the above cell adhesion and proliferationmethod, which comprises inoculating, culturing and then killingfibroblast derived from a mammal to provide improved cell adhesion andproliferation. However, complete removal of fibroblasts may not alwaysbe necessary in the aspect of cell adhesion and proliferation.Accordingly, the dead cells may be removed at least partially such thatcell adhesion or proliferation may not be inhibited. The term “beseparated at least partially” herein, which refers to the extent ofremoval of dead cells, particularly means removing preferably 50% ormore, more preferably 80% or more, and most preferably 100% of deadcells when compared to the total amount of viable fibroblasts justbefore killed.

Dead cells may be generally removed by rinsing the culture surface withany isotonic solution which does not degenerate the active component orcomponents required for cell adhesion and proliferation, such asphosphate buffer, Hanks' solution and saline.

The extent to which dead cells are removed can be easily confirmed byusing, for example, a phase contrast microscope. When 100% removal ofdead cells is confirmed, then it means that the resultant epidermal cellsheet, epidermal cell suspension or hepatic cells are free ofheterogenous cells such as 3T3 mouse embryo fibroblast. It is notdifficult to remove 100% of dead cells. Dead cells can be easily removedalmost completely by any conventional procedure. Alternatively, deadcells will completely be apart from the surface of the vessel into theculture solution. Therefore, an epidermal cell sheet, an epidermal cellsuspension or hepatic cells obtained by using the culture vesselaccording to the present invention which can provide improved adhesionto cell and enhanced cell-proliferation may be substantially free of anyheterogenous cell. Conventionally, a practical epidermal cell sheetcould be prepared only be feeder layer culture method which may causecontamination with heterogeneous cells to some extent when cells derivedfrom a mammal other than human are used as the feeder cells. On theother hand, an epithelial cell sheet, i.e., a muscosa epithelial cellsheet or a muscosa epithelial cell suspension, or an epidermal cellsheet or an epidermal cell suspension obtained by using the culturevessel according to the present invention which can provide improvedadhesion to cell and enhanced cell-proliferation are free ofheterogeneous cells.

The culture vessel which can provide improved adhesion to cell andenhanced cell-proliferation (e.g., a petri dish containing component(s)required for adhesion and proliferation of the target cell from thesurface of which dead cells have been removed) can be left to stand, forexample, in a refrigerator at 2° C. to 8° C. or in a deep freezer at−30° C., −80° C. or the like for at least about 0.5 to 1 year whilekeeping its property. This can eliminate several steps such as preparinga feeder layer just before inoculating of epidermal cells, deletingdivision potency of, for example, 3T3 fibroblasts by irradiating γ-rayor by adding mitomycin C and thereafter inoculating the cells (1×10⁴cells/cm²). Further, conventional feeder layers could retain theirproperty only for about 2 days. On the other hand, the culture vesselaccording to the present invention, which can provide improved adhesionto cell and enhanced cell-proliferation, can be preserved for about 0.5to 1 year while retaining such property. Moreover, a large number of theculture vessel according to the present invention can be easilymanufactured at one time, and they can provide similar activity. Thusthe process costs can be greatly reduced. Additionally, the methodaccording to the present invention does not require daily subculture offibroblasts for preparation of feeder layer.

The above epidermal cell sheet can be easily prepared in a desiredamount when required, by using the culture vessel according to thepresent invention. Particularly such an epidermal cell sheet may beprepared by a method similar to any conventional process for preparingepidermal cell sheet except for using the culture vessel according tothe present invention. For example, epidermal cells are inoculated(1.0×10⁴ cells/cm²) and then cultured for about 7 to 21 days whilechanging the medium about twice a week to prepare an epidermal cellsheet.

The above epidermal cell suspension can be prepared by any conventionalmethod for preparing epidermal cell suspension except for using theculture vessel according to the present invention. For example,epidermal cells are inoculated (1.0×10⁴ cells/cm²) and then cultured forabout 3 to 21 days while changing the medium about twice a week.Thereafter, the cells are treated with an enzyme such as trypsin tosubstantially obtain single cells which are then suspended in a solutionsuch as neutral collagen solution.

Hereinafter, the present invention will be explained by way of followingexamples. These examples are not intended to limit the scope of thepresent invention.

EXAMPLE Example 1

Established 3T3 mouse embryo fibroblasts were inoculated in a cultureflask (culture surface: 25 cm²) at 3×10³ cells/cm² and incubated in aCO₂ incubator (at 37° C., 5% CO₂) for 4 days. The medium was Dulbecco'smodified Eagle medium supplemented with 10% fetal bovine serum(DMEM+10%FBS).

After incubation, culture supernatant in the culture flask was removedby aspiration, and the remainder was left to stand in a deep freezer at−85° C. for 12 hours for freezing. Next, the frozen culture was thawedat room temperature, and the culture surface was rinsed with 5 ml ofphosphate buffer to remove dead cells. Then, the culture flask was againfrozen by leaving it to stand in the deep freezer at −85° C. overnight.

The culture flask (hereinafter referred to as “3T3 frozen”) was thawedat room temperature and inoculated with epidermal cells (1×10⁴cells/cm²) collected from human skin.

On the other hand, epidermal cells were inoculated at 1×10⁴ cells/cm² ina culture flask (culture surface; 25 cm²) containing a feeder layerconsisting of 3T3 mouse embryo fibroblasts which have been treated withmitomycin C to delete their division potency as a control.

These culture flasks were incubated in a CO₂ incubator (37° C. 5% CO₂)for 8 days. The medium was Green medium supplemented with 3% fetalbovine serum (Green+3%FBS).

After 8-day incubation, an epidermal cell sheet was prepared on thesurface of each culture vessel. The sheet was separated from the culturesurface of each culture vessel by treating with dispase, the epidermalcells were treated with trypsin solution (which was prepared bydissolving trypsin (0.25 weight/volume %) in a solution of 0.206 mg/mlethylenediamine-tetraacetic acid (EDTA) in phosphate buffer) to obtainsingle cells, and the cells were counted on a hemacytometer.

The dispase treatment was performed by dissolving 10,000PU dispase in 10ml of Dulbecco's modified Eagle medium to prepare dispase solution,adding 3 ml of the dispase solution to the culture flask, and leavingthe flask to stand in a CO₂ incubator for about 1 hour.

Epidermal cells were counted for both the control and 3T3 frozen intriplet, and the average of the results were shown in FIG. 1 forcomparison. As shown in FIG. 1, more epidermal cells were adhered andproliferated in the culture vessel according to the present invention(3T3 frozen) when compared to those grown by inoculating epidermal cellsaccording to the conventional feed layer culture method (control), whichemployed 3T3 mouse embryo fibroblast as feeder cell, and incubating for8 days.

Example 2

Established 3T3 mouse embryo fibroblasts were inoculated in a cultureflask (culture surface: 25 cm²) at 3×10³ cells/cm² and incubated in aCO₂ incubator (at 37° C., 5% CO₂) for 4 days. The medium was Dulbecco'smodified Eagle medium supplemented with 10% fetal bovine serum(DMEM+10%FBS).

After incubation, culture supernatant in the culture flask was removedby aspiration, and the remainder was freeze-dried in a freeze dryer.Schedule used was as follows: retaining at −30° C. for 1 hour; drying byvacuum aspiration; heating at 1.5° C./minute; and retaining at 20° C.for 20 hours.

Epidermal cells collected from human skin were inoculated in the cultureflask at 1×10⁴ cell/cm². The medium was Green medium supplemented with3% fetal bovine serum. After 4 days, the colonies of epidermal cellswere observed. The epidermal cells were proliferated withoutcontamination with 3T3 mouse embryo fibroblasts.

Example 3

Established 3T3 mouse embryo fibroblasts were inoculated in a cultureflask (culture surface: 25 cm²) at 3×10³ cells/cm² and incubated in aCO₂ incubator (at 37° C., 5% CO₂) for 4 days. The medium was Dulbecco'smodified Eagle medium supplemented with 10% fetal bovine serum(DMEM+10%FBS).

After incubation, culture supernatant in the culture flask was removedby aspiration, and the remainder was left to stand in a deep freezer for12 hours for freezing. Next, the frozen culture was thawed at roomtemperature, and the culture surface was rinsed with 5 ml of phosphatebuffer to remove dead cells. Then, the culture flask was again frozen byleaving it to stand in the deep freezer at −85° C. overnight.

The culture flask (3T3 frozen) was thawed at room temperature andinoculated with epidermal cells collected from human skin at 1×10⁴cells/cm².

These culture flasks were incubated in a CO₂ incubator (37° C., 5% CO₂)for 8 days. The medium was Green medium supplemented with 3% fetalbovine serum (Green+3%FBS).

After 8-day incubation, epidermal cells were separated from the culturesurface of each culture vessel, treated with trypsin solution (which wasprepared by dissolving trypsin (0.25 weight/volume %) in a solution of0.206 mg/ml ethylenediamine-tetraacetic acid (EDTA) in phosphate buffer)to obtain single cells, and then the cells were counted on ahemacytometer. 3×10⁵ cells/ml epidermal cell suspension was prepared bysuspending cells in a neutral collagen solution (which was prepared bydissolving aterocollagen derived from a hog (0.2 weight/volume %) inDulbecco's modified Eagle medium, and by adjusting at pH 7.4).

Example 4

Established 3T3 mouse embryo fibroblasts wee inoculated in a cultureflask (culture surface: 80 cm²) at 3×10³ cells/cm² and incubated in aCO₂ incubator (at 37° C., 5% CO₂) for 3 days. The medium was Dulbecco'smodified Eagle medium supplemented with 10% fetal bovine serum(DMEM+10%FBS).

After incubation, culture supernatant in the culture flask was removedby aspiration, and then the remainder was treated by irradiating 10kGy-and 25kGy- electron beam at 4° C.

Epidermal cells collected from human skin were inoculated in the cultureflask at 1×10⁴ cell/cm². The medium was Green medium supplemented was 3%fetal bovine serum.

After that, epidermal cells were adhered and proliferated in the culturevessel. The epidermal cell sheet could be prepared. 3T3 fibroblasts wereobserved to proliferate in the culture vessel which had not been treatedby irradiating electron beam, but not observed in the culture vesseltreated by irradiating electron beam.

What is claimed is:
 1. A method for adhering and proliferatingepithelial cells, which comprises the steps of: inoculating, culturingand then treating by a treatment selected from freezing, drying andirradiating, in a culture vessel, fibroblasts derived from a mammal,separating the treated fibroblasts from said culture vessel on which thefibroblasts were cultured and treated, so as to leave an extracellularmatrix on a surface of said culture vessel, and then inoculating andculturing the epithelial cell in said culture vessel.
 2. The methodaccording to claim 1, wherein 50% or more of the treated fibroblasts areseparated from the culture vessel.
 3. The method according to claim 1,wherein the treated fibroblasts are separated from the culture vesselentirely.
 4. The method according to claim 1, in which said epithelialcells are epidermal cells.
 5. The method according to claim 1, whereinsaid fibroblasts are treated by at least one selected from the groupconsisting of β ray, γ ray, X-ray, electron beam and UV ray.
 6. Themethod according to claim 1, wherein said treating step of fibroblastscomprises repeating one treatment selected from the group consisting offreezing, drying and irradiating.
 7. The method according to claim 1,wherein said treating step of fibroblasts comprises repeating exposureto at least one selected from the group consisting of β ray, γ ray,electron beam, UV ray and X-ray.
 8. The method according to claim 1,wherein said fibroblasts are treated by a combination of at least twotreatments selected from the group consisting of freezing, drying andirradiating.
 9. The method according to claim 1, wherein fibroblasts aretreated by at least one selected from the group consisting of β ray, γray, electron beam, UV ray and X-ray.
 10. The method according to claim1, wherein said fibroblasts are 3T3 mouse embryo fibroblasts.
 11. Themethod according to claim 1, wherein the treated fibroblasts areseparated entirely from said culture vessel by rinsing the culturesurface with an isotonic solution.